Management of chronic wounds represent a major healthcare challenge responsible for over $15 billion in expenses annually. Wound surface sepsis, in particular biofilm formation, is a significant factor in nonhealing wounds. In chronic wounds, >60% have been observed to have clear evidence of a biofilm. Further, NIH has estimated that 65-80% of microbial infections in humans are biofilm-mediated. Biofilms are characterized by resistance to host defenses and to therapeutics that would otherwise have efficacy against the organisms planktonic state. Thus, biofilms present two distinct challenges: 1) the biofilm must be removed or dispersed to reduce bacterial defense mechanisms; and 2) effective antimicrobial agents applied to suppress the resident bacteria. In this application, we seek to address this problem by investigating a novel intervention with potential to prevent chronic colonization of wounds and disperse pre-existing biofilms. The innovation underlying our approach revolves around engineering the surfaces of wound beds to both promote the dissolution of biofilm bacteria back to a planktonic state, where they are more susceptible to antimicrobial agents, and immobilize antimicrobial agents at the wound surface where biofilm formation occurs. Our approach involves incorporation of absorbable nanobeads loaded with antibiofilm agents in a nanoscopic thin film, manufactured of polyelectrolyte multilayers containing silver nanoparticles, that is used to re-engineer the wound surface to increase its resistance to microbial colonization and biofilm formation. This structured engineered approach allows effective antimicrobial action with very low non-toxic concentrations of active agents. The innovative combination of these approaches in a single nanostructured film has the potential to markedly increase anti-biofilm and antimicrobial efficacy in vivo. In particular, use of nano-beads permits precise control over concentrations and release rates of the antibiofilm agent. They also penetrate and create microdomains within the biofilm, increasing surface contact by >80%, to increase adsorption of antibiofilm and antimicrobial agents concurrently and generating progressive dispersion-kill zones emanating from the beads at a nanoscale level. The central hypothesis of this study, supported by exciting preliminary data, is that incorporating select D- and L- amino acids into the wound bed will reduce bacterial biofilm formation and increase biofilm dissolution. A secondary hypothesis is that this approach will enhance the antimicrobial activity of silver nanoparticles immobilized at the wound surface. To address these goals we propose 3 Aims. In Aim 1, we will evaluate the ability of select D- and L- amino acids immobilized in polyelectrolyte thin films and loaded into absorbable PLGA beads to prevent a biofilm from forming and to stimulate dissolution of existing biofilms of Pseudomonas aeruginosa and Staphylococcus aureus in vitro . In Aim 2, we will optimize the integration of select amino acids and nano-beads onto model wound surfaces (full and partial thickness skin wounds), using polyelectrolyte thin film immobilization methods, and evaluate their efficacy in preventing biofilm formation and biofilm dissolution in vivo; and in Aim 3, we will test the hypothesis that combined application of silver nanoparticles and select amino acids further reduces biofilm formation and minimizes microbial bioburden in wounds in vivo. At the conclusion of this study, we expect to provide proof of concept that a two-armed approach to wound biofilms contained in an integrated nanoscale wound bed engineering platform will have increased efficacy against biofilms with reduced cytotoxicity in the wound bed. This approach is labile and generalizable to immobilization of other antibiofilm and antimicrobial agents. Thus, upon successful completion of this R21 application, we will seek support for research (via the R01 mechanism) that will broaden the scope of these investigations in optimizing these strategies and in evaluating the efficacy of an array of antimicrobial and antibiofilm agents with the goal of maximizing the ability to suppress wound bed sepsis and improve healing of chronic open wounds. PUBLIC HEALTH RELEVANCE: This application seeks support for an exploratory investigation of a conceptually novel approach to suppression of wound microbial bioburden and biofilm formation. Simple amino acids have been shown to be effective in preventing biofilms from forming and in promoting biofilm dissolution. These compounds, when integrated into a nanostructured wound surface engineering platform, offer a two-armed approach to dissolution of biofilms and killing of resident bacteria by their combination with a non-antibiotic antimicrobil such as silver in nanoparticulate form. The engineered immobilization platform allows use of low non-toxic concentrations of active agents. Such an approach provides a highly innovative attack of a major health problem without the side effects of cytotoxicity or microbial antibiotic resistance inherent to current conventional wound treatments. We seek to test this hypothesis using a wound engineering approach involving polyelectrolyte multilayers with absorbable nanobeads to deliver both antibiofilm and antimicrobial agents together at the wound surface. Approaches such as this that facilitate prevention of wound sepsis that do not impair wound healing would represent a major healthcare advance.